Institute for Reference Materials and Measurements (IRMM) Joint Research Centre Institute for Reference Materials and Measurements (IRMM) http://irmm.jrc.ec.europa.eu

Abeta42 standardisation Definition of the SI unit e.g. AAA, refractive index “Pure” peptide calibrator Reference procedure, immunoassay ring trial CRM matrix material Value transfer 1: LC-MS, immunoassay Manufacturer’s working calibrator Value transfer 2: immunoassay Manufacturer’s product calibrator Immunoassay Routine sample Aim: Setting up a reliable traceability chain linking results on routine samples to a common reference, the international system of units (SI) 2

✔ ✔ Definition of the SI unit Purity assessment calibrant Purity assessment amino acids ✔ Pure amino acids Amino acid analysis validation ✔ Amino acid analysis Abeta42 calibrant Commutability studies Correlation studies LC-MS Reference procedure CRM matrix material Processing LC-MS ring trial Feasibility studies processing

Calibrator batch: - Intended use: Calibration of LC-MS measurements for the value assignment of the CRM - Format: recombinant Abeta42 in 20 % acetonitrile, 1 % ammoniumhydroxide - Concentration: determined by Amino acid analysis and purity assessment

Calibrator batch: Amino Acid analysis: - Problem in-house with repeatability and between AA variation, indicating a problem.

Possible explanations: - Purity: very high purity (Q-TOF, ion-trap, sequencing) - Digestion conditions optimised - Different sequences present? Unlikely as recombinant, confirmed by MS sequencing - Problematic interaction between solvent and HCl → freezedrying of samples before measurements: good conditions found on a different test batch To be repeated on actual calibrant (ongoing)

✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit Purity assessment calibrant Purity assessment amino acids ✔ Pure amino acids Amino acid analysis validation ✔ ✔ Amino acid analysis ✔ Abeta42 calibrant ✔ Commutability studies Correlation studies LC-MS Reference procedure CRM matrix material Processing LC-MS ring trial Feasibility studies processing

LC-MS reference method Must give results that correlate with routine assays → correlation studies: OK Pannee et al (2013) J Alzheim Dis Leinenbach et al (2014) Clin Chem Korecka et al (2014) J Alzheim Dis Bjerke et al (2015) CCLM submitted

Ring trial with common protocol for calibration 9 % 6 datasets: Uncertainty for value assignment:   Acceptable for value assignment of the CRM !

✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit Purity assessment calibrant ✔ Purity assessment amino acids ✔ Pure amino acids Amino acid analysis validation ✔ ✔ Amino acid analysis ✔ Abeta42 calibrant ✔ Commutability studies Correlation studies ✔ LC-MS Reference procedure ✔ CRM matrix material Processing LC-MS ring trial ✔ Feasibility studies processing

Commutability: a reference material must behave in the same way as a patient sample (2 studies, 10 methods) Bjerke et al, CCLM submitted

Processing tests: - Manual filling/ machine filling - Stirring/ not stirring (no difference) - Abeta42 concentration over time while stirring (no slope) - Flash freezing versus slow freezing (better slow freezing) Drift according to filling sequence (aCSF) problem at beginning and end of filling (aCSF)

Conditions chosen: - Machine filling - Stirring - slow freezing Statistical evaluation of data: Soverall = 3.8 % Srepeatability = 3.2 % (method variability) Sbetween vial = 1.9 % (between vial variability) Results from last test (CSF)

→ Decision on format of the matrix reference material: - CSF pool - No spiking - 0.5 mL/vial - 3 levels: low, medium and high - Stored at -70°C

1st level filled

✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Definition of the SI unit Purity assessment calibrant ✔ Purity assessment amino acids ✔ Pure amino acids Amino acid analysis validation ✔ ✔ Amino acid analysis ✔ Abeta42 calibrant ✔ Commutability studies ✔ Correlation studies ✔ LC-MS Reference procedure ✔ ✔ CRM matrix material Processing ✔ LC-MS ring trial ✔ Feasibility studies processing ✔

CRM Distribution and Sales Planning Feasibility study (collection, processing, characterisation, commutability) Correlation studies Homogeneity study Storage Processing Interim transport and storage Material selection Short-term stability study Long-term stability study Value assignment Commutability study Follow-up stability monitoring of CRM Assessment by experts Documentation CRM Distribution and Sales

→ Next step 1: homogeneity study first material → Next step 2: processing 2 other levels → Next step 3: Value assignment: - LC-MS methods validated - 6 laboratories or methods - 3 levels - At least 2 days - Common protocol for calibration

Thanks to a large collaboratve network: GBSC IFCC Participating laboratories Manufacturers Some people devoting a majority of their time: Josef Pannee, Julia Kuhlmann, Maria Bjerke, Magdalena Korecka, Andreas Leinenbach…